1. Field of the Invention
The present invention relates to a method and apparatus for controlling the temperature of a liquid and in particular to a method and apparatus for controlling the temperature of a liquid in a thermal cycling apparatus. The present invention also relates to a method and apparatus for determining the temperature of a liquid in a thermal cycling apparatus.
2. Description of Related Art
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
PCR (Polymerase Chain Reaction) is a technique involving multiple cycles that results in the exponential amplification of certain polynucleotide sequences each time a cycle is completed. The technique of PCR is well known and is described in many books, including, PCR: A Practical Approach M. J. McPherson, et al., IRL Press (1991), PCR Protocols: A Guide to Methods and Applications by Innis, et al., Academic Press (1990), and PCR Technology: Principals and Applications for DNA Amplification H. A. Erlich, Stockton Press (1989). PCR is also described in many U.S. patents, including U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; 4,889,818; 5,075,216; 5,079,352; 5,104,792; 5,023,171; 5,091,310; and 5,066,584.
The PCR technique typically involves the step of denaturing a polynucleotide, followed by the step of annealing at least a pair of primer oligonucleotides to the denatured polynucleotide, i.e., hybridizing the primer to the denatured polynucleotide template. After the annealing step, an enzyme with polymerase activity catalyzes synthesis of a new polynucleotide strand that incorporates the primer oligonucleotide and uses the original denatured polynucleotide as a synthesis template. This series of steps (denaturation, primer annealing, and primer extension) constitutes a PCR cycle.
As cycles are repeated, the amount of newly synthesized polynucleotide increases exponentially because the newly synthesized polynucleotides from an earlier cycle can serve as templates for synthesis in subsequent cycles. Primer oligonucleotides are typically selected in pairs that can anneal to opposite strands of a given double-stranded polynucleotide sequence so that the region between the two annealing sites is amplified.
Denaturation of DNA typically takes place at around 90 to 95° C., annealing a primer to the denatured DNA is typically performed at around 40 to 60° C., and the step of extending the annealed primers with a polymerase is typically performed at around 70 to 75° C. Therefore, during a PCR cycle the temperature of the reaction mixture must be varied, and varied many times during a multicycle PCR experiment.
The PCR technique has a wide variety of biological applications, including for example, DNA sequence analysis, probe generation, cloning of nucleic acid sequences, site-directed mutagenesis, detection of genetic mutations, diagnoses of viral infections, molecular “fingerprinting” and the monitoring of contaminating microorganisms in biological fluids and other sources.
In addition to PCR, other in vitro amplification procedures, including ligase chain reaction as disclosed in U.S. Pat. No. 4,988,617 to Landegren and Hood, are known and advantageously used in the prior art. More generally, several important methods known in the biotechnology arts, such as nucleic acid hybridization and sequencing, are dependent upon changing the temperature of solutions containing sample molecules in a controlled fashion. Conventional techniques rely on use of individual wells or tubes cycled through different temperature zones. For example, a number of thermal “cyclers” used for DNA amplification and sequencing are disclosed in the prior art in which a temperature controlled element or “block” holds a reaction mixture, and wherein the temperature of the block is varied over time. One advantage of these devices is that a relatively large number of samples can be processed simultaneously, e.g. 96 well plates are commonly employed.
U.S. Pat. No. 7,645,070 describes an instrument for performing highly accurate PCR employing an assembly, a heated cover, and an internal computer, is provided. The assembly is made up of a sample block, a number of Peltier thermal electric devices, and a heat sink, clamped together. A control algorithm manipulates the current supplied to thermoelectric coolers such that the dynamic thermal performance of a block can be controlled so that pre-defined thermal profiles of sample temperature can be executed. The sample temperature is calculated instead of measured using a design specific model and equations. The control software includes calibration diagnostics which permit variation in the performance of thermoelectric coolers from instrument to instrument to be compensated for such that all instruments perform identically. The block/heat sink assembly can be changed to another of the same or different design. The assembly carries the necessary information required to characterize its own performance in an on-board memory device, allowing the assembly to be interchangeable among instruments while retaining its precision operating characteristics.
U.S. Pat. No. 5,475,610 describes an instrument for performing highly accurate PCR employing a sample block in microtiter tray format. The sample block has local balance and local symmetry. A three zone film heater controlled by a computer and ramp cooling solenoid valves also controlled by the computer for gating coolant flow through the block controls the block temperature. Constant bias cooling is used for small changes. Sample temperature is calculated instead of measured. A platen deforms plastic caps to apply a minimum acceptable threshold force for seating the tubes and thermally isolates them. A cover isolates the block. The control software includes diagnostics. An install program tests and characterizes the instrument. A new user interface is used. Disposable, multipiece plastic microtiter trays to give individual freedom to sample tubes are taught.
However, such block devices suffer various drawbacks, in that they are relatively slow in cycling the reaction mixtures, they are relatively energy intensive to operate, temperature control is less than ideal and detection of the reaction mixture in situ is difficult.
In an effort to avoid several of these disadvantages, other thermal cyclers have been developed in which a plurality of containers for holding reaction mixture(s) are supported on a rotatable carousel rotatably mounted within a chamber adapted to be heated and cooled. For example, see U.S. Pat. No. 7,081,226 to Wittwer et al. However, these devices still suffer various disadvantages. For example, control over the temperature of the reaction mixtures is less than ideal, control over the rate of heating and cooling of the reaction mixtures is less than ideal, and these devices have relatively poor energy efficiency.
Thus, there still remains a need for thermocyclers for PCR which provide improved temperature control of the reaction mixtures, are not complex to use, can provide real-time analysis of the reaction occurring in the sample containers, and are energy efficient.